The objectives of the proposed research are to elucidate the mechanism of methemoglobin reduction as it occurs in erythrocytes, to probe the role of proteases in the origin of the components of the methemoglobin reduction system, and to establish the structre of the prosthetic group and the biological activity of an erythrocyte green hemeprotein. We will compare the chemical structures and physical properties of the two forms of bovine erythrocyte cytochrome b5. The proteins will be compared in terms of UV, vivible, CD, MCD, and ORD spectra, and amino acid sequence at the C-terminus. The nature of the blocked N-terminal residues will be investigated. We will establish the structural relationship between the liver microsomal and erythrocyte cytochrome b5. We will attempt to isolate and characterize proteases which degrade microsomes to yield form I or II of the erythrocyte cytochrome b5. We will solubilize cytochrome b5 redctase from microsomes with these same proteases and compare the resulting solubilized forms of reductase with the soluble cytochrome b5 reductase of erythrocytes. The kinetics of methemoglobin reduction will be studied in a system reconstituted from purified cytochrome b5, cytochrome b5 reductase, methemoglobin, and NADH, in erythrocyte hemolysates, and in intact cells. We will study the system in erythrocytes from normal individuals and from patients with congenital methemoglobinemia; the amount of cytochrome b5 in these cells will be measured. The mechanism of electron transfer will be studied in partial reactions of the system with purified proteins. We will determine the molecular weight of the prosthetic group of the bovine erythrocyte green hemeprotein, derivatize this heme, and characterize these derivatives spectrally. We will search for the biological activity of this protein.